The objective of this research project is to elucidate the mechanism(s) which underlies the association between carcinogenesis and cell proliferation. The principal focus of the project is on a newly reported method for purification of eukaryotic replication complexes from the nuclei of proliferating rat liver cells. The proposed studies will utilize this method to elucidate the process of eukaryotic DNA replication and to characterize the effect of carcinogens on biologically determined DNA subfractions. Proposed studies will further characterize the constituents of the replication complex. The generality of the procedure for isolating replication complexes will be determined using other replicating cells. The observation that replication complexes associated with nuclear membrane show the greatest increase in synthesis of DNA at the earliest stages of the S-phase indicates a possible method for identification of S-phase initiation sites. The generality of this observation will be tested in a variety of well-synchronized proliferating cells. The observation of preferential binding of a chemical carcinogen to DNA at the replication complex will be further examined with various carcinogenic and non-carcinogenic (or weakly carcinogenic) alkylating agents. Alkylation of nascent and parental DNA strands at the replication site will be evaluated. The nucleotide sites of alkylation will be compared for DNA at and away from the replication complex. Since nuclear membrane associated replication complexes at the time of the G1-S transition appear likely to be associated with S-phase initiation sites, alkylation of this subfraction of DNA will be considered. Experiments will determine whether this fraction is preferentially alkylated by carcinogenic doses and whether this preferential alkylation is lost at higher toxic doses.